Solid-Phase Primer Elongation Using Biotinylated dNTPs for the Detection of a Single Nucleotide Polymorphism from a Fingerprick Blood Sample
| Autor: | Miriam Jauset-Rubio, Ciara K. O'Sullivan, Mayreli Ortiz |
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| Rok vydání: | 2021 |
| Předmět: |
Letter
Chromatography Nucleotides Chemistry 010401 analytical chemistry Recombinase Polymerase Amplification DNA 02 engineering and technology Ion semiconductor sequencing 021001 nanoscience & nanotechnology Polymorphism Single Nucleotide 01 natural sciences DNA sequencing Primer extension 0104 chemical sciences Analytical Chemistry chemistry.chemical_compound genomic DNA Genetic Techniques Biotinylation Primer (molecular biology) 0210 nano-technology DNA Primers |
| Zdroj: | Analytical Chemistry |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.1c03419 |
| Popis: | Isothermal recombinase polymerase amplification-based solid-phase primer extension is used for the optical detection of a hypertrophic cardiomyopathy associated single nucleotide polymorphism (SNP) in a fingerprick blood sample. The assay exploits four thiolated primers which have the same sequences with the exception of the 3'-terminal base. Target DNA containing the SNP site hybridizes to all four of the immobilized probes, with primer extension only taking place from the primer containing the terminal base that is complementary to the SNP under interrogation. Biotinylated deoxynucleotide triphosphates are used in the primer extension, allowing postextension addition of streptavidin-poly-horseradish peroxidase to bind to the incorporated biotinylated dNTPs. The signal generated following substrate addition can then be measured optically. The percentage of biotinylated dNTPs and the duration of primer extension is optimized and the system applied to the identification of a SNP in a fingerprick blood sample. A methodology of thermal lysis using a 1 in 5 dilution of the fingerprick blood sample prior to application of 95 °C for 30 s is used to extract genomic DNA, which is directly used as a template for solid-phase primer extension on microtiter plates, followed by optical detection. The SNP in the fingerprick sample was identified and its identity corroborated using ion torrent next generation sequencing. Ongoing work is focused on extension to the multiplexed detection of SNPs in fingerprick and other biological samples. |
| Databáze: | OpenAIRE |
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